395 395 395 395 395 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 92(3): 395-398, May/Jun. 1997
A Simple Reverse Transcription-Polymerase Chain Reaction
for Dengue Type 2 Virus Identification
Luiz Tadeu M Figueiredo/
+, Weber Chelli Batista, Akira Igarashi
*Unidade Multidisciplinar de Pesquisa em Virologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, 14049-900 Ribeirão Preto, SP, Brasil *Department of Virology, Institute of Tropical Medicine,
Nagasaki University, 12-4 Sakamoto-machi, Nagasaki, Japan
We show here a simplified reverse transcription-polymerase chain reaction (RT-PCR) for identi-fication of dengue type 2 virus. Three dengue type 2 virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD, as a negative control, were used in this study. C6/36 cells were infected with the virus, and tissue culture fluids were collected after 7 days of infection period. The RT-PCR, a combination of RT and PCR done after a single addition of reagents in a single reaction vessel was carried out following a digestion of virus with 1% Nonidet P-40. The 50 µl assay reaction mixture included 50 pmol of a dengue type 2 specific primer pair amplifying a 210 base pair sequence of the envelope protein gene, 0.1 mM of the four deoxynucleoside triphosphates, 7.5U of reverse transcriptase, and 1U of thermostable Taq DNA polymerase. The reagent mixture was incubated for 15 min at 37oC for RT followed by a variable amount of cycles of two-step PCR amplification (92oC for 60 sec, 53oC for 60 sec) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel elec-trophoresis and visualized with UV light after gel incubation in ethidium bromide solution. DNA bands were observed after 25 and 30 cycles of PCR. Virus amount as low as 102.8 TCID50/ml was detected by RT-PCR. Specific DNA amplification was observed with the three dengue type 2 strains. This assay has advantages compared to other RT-PCRs: it avoids laborious extraction of virus RNA; the combination of RT and PCR reduces assay time, facilitates the performance and reduces risk of contamination; the two-step PCR cycle produces a clear DNA amplification, saves assay time and simplifies the technique.
Key words: dengue identification - dengue diagnosis - reverse transcription-polymerase chain reaction
The four dengue virus serotypes, designated 1, 2, 3, and 4, are positive sense, single-stranded RNA viruses belonging to the genus Flavivirus of the family Flaviviridae (WHO 1986). According to strict epidemiological criteria, dengue viruses are arboviruses (arthropod-borne viruses) transmitted by Aedes aegypti and Ae. albopictus mosquitoes (Wengler 1991). These viruses cause an impor-tant arboviral disease of man, dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) (Gubler 1987).
Since the mid-80s, dengue epidemics have oc-curred throughout Brazil, producing millions of infections. Dengue types 1 and 2 have been iso-lated during epidemics. The vast majority of the disease cases involved were not life-threatening. However, after the introduction of dengue type 2, hundreds of DHF/DSS cases have occurred in the states of Rio de Janeiro and Ceará causing several deaths (Figueiredo et al. 1995).
Research supported by Japan International Cooperation Agency, and Brazilian Agencies CNPq and CAPES. +Corresponding author. Fax: +55-16-633.6695 Received 28 November 1996
Accepted 3 February 1997
The identification of Brazilian dengue virus isolates has been done by using type-specific mono-clonal antibodies in an immunofluorescence test utilizing infected mosquito cells fixed on micro-scope slides (Henchal et al. 1983). Reverse tran-scription (RT) followed by polymerase chain re-action (PCR) has been used for rapid and specific detection of flaviviruses including the dengue vi-ruses (Shibata et al. 1990, Laille et al. 1991, Lanciotti et al. 1992, Morita et al. 1994, Beaty et al. 1995). We show here a simplified RT-PCR for detection and identification of dengue type 2 vi-rus.
MATERIALS AND METHODS
Virus strains - The three dengue type 2 virus
strains, CEA-2462, SPH125367 and TOC-213 (Table I), used in this study, were isolated from Brazilian patients, and kindly supplied by Dr Amélia Travassos da Rosa from Evandro Chagas Institute, Brazilian Health Ministry, Belém, State of Pará, and Dr Luiza Teresinha Madia de Souza from Adolpho Lutz Institute, State of São Paulo Health Ministry. Yellow fever vaccine virus 17DD (Fiocruz, Brazil) was used as a negative control.
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C6/36 (Ae. albopictus) cells were grown at 28oC in Leibovitz L-15 medium containing 6% heat-inac-tivated fetal bovine serum, 10% tryptose phosphate broth, 100 U/ml penicillin and 100 µg/ml streptomy-cin. The cells were infected with virus strains, and tissue culture fluids were collected as virus seeds after 7 days of infection with confirmation of the infection by indirect immunofluorescence (Henchal et al. 1983, Figueiredo 1990). Infected tis-sue culture fluids were stored at -70oC, and ten-fold dilutions were prepared in tissue culture me-dium at time of the assay.
TABLE I Dengue type 2 virus strains
Strain Place of infection Year
DEN 2 CEA 2462 Ceará 1994
DEN 2 SPH 125367 Rio de Janeiro 1991
DEN 2 TOC 213 Tocantins 1991
Titration of dengue type 2 CEA 2462 - C6/36
cells were added to 96-well cluster dishes (Corn-ing, USA) in 100 µl of tissue culture medium at a density of 2x104 cells per well. After 24 hr the cells were infected with 50 µl of tenfold dilutions of den-gue type 2 CEA 2462, starting from 10-0 to 10-5, with 6 wells for each virus dilution. After a 5 day incubation period, all the wells received 50 µl of neutral buffered formalin (pH 7) and were held overnight at 4oC.
An enzyme immunoassay was performed after washing the microplate wells twice in phosphate buffered saline (PBS; Gibco BRL, USA). 100 µl of dengue type 2 mouse polyclonal antiserum diluted 1/100 in PBS containing 0.5% bovine serum albu-min (BSA) was added to the wells. After 1 hr at 37oC and 3 washes with PBS, horseradish peroxi-dase-conjugated goat anti-mouse IgG (Sigma, USA) diluted 1/2000 in 0.5% BSA-PBS was added to the wells. Microplates were maintained for 1 hr at 37oC and washed 5 times in PBS. Results were read spectrophotometrically (414 nm), 30 min af-ter the addition of ABTS substrate (Kirkegaard and Perry, USA) to the microplate wells (Figueiredo & Shope 1987).
Infected wells were determinated as positives when their OD exceeded 3 SD of the mean value for control wells in the assay. The virus titer (TCID50) was calculated by the method of Reed and Muench (1938).
Primers - Dengue type 2 specific primers
am-plifying a 210 base pair sequence of the envelope protein gene, were synthesized by Gibco BRL (USA) (Deubel et al. 1988, Morita et al. 1993, Tanaka 1993). Primer sequences and position in the virus genome are shown in Table II.
TABLE II
Nucleotide sequences and genome position of dengue primers
Code Sequence Position
DEN2-S GTT CCT CTG CAA ACA CTC CA 1203-1222 DEN2-C GTG TTA TTT TGA TTT CCT TG 1432-1413
S: sense primer; C: complementary primer
RT-PCR - The test was done in 500 µl Eppendorf tubes by adding 1 µl of infected tissue culture fluid (different dilutions were used), and 5 µl of a deter-gent mixture containing 1% Nonidet P-40 and 5 U of RNase inhibitor (Pharmacia, USA) in PBS. After 60 sec of incubation at room temperature for virus solubilization, the cDNA was added to a reaction mixture containing 50 pmol of the primer pair, 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase (Pharmacia, USA), 1U of Taq DNA polymerase (Pharmacia, USA), and 5 µl of a buffer solution containing 5 mM Tris (pH 9.0), 0.75 mM MgCl2, and 25 mM KCl. A total volume of 50 µl was obtained by adding distilled and deion-ized water. The reaction mixture was covered with 2 drops of oil and was incubated for 15 min at 37oC for RT, followed by 15 to 30 cycles of a two step PCR amplification (92oC for 60 sec, 53oC for 60 sec) in a thermal cycler (Techne, UK). The tem-perature increment was slow, taking 120 sec from 53oC to 92oC, and 15 sec from 70oC to 75oC. The total time for the test was 150 min.
Eight µl of each PCR product was subjected to electrophoresis in a 1.7% agarose gel. PCR prod-uct nucleotide was visualized in UV light after in-cubation of the gel in a 0.5 µg/ml ethidium bromide solution for 20 min, and rinsing with tap water. The size of the amplified DNA fragments was determined by comparing their migrations in the gel with those of the bands of a 100 base pair molecular weight marker (Pharmacia, USA).
RESULTS
The undiluted dengue type 2 CEA 2462 seed was processed at 20, 25 and 30 cycles of PCR af-ter RT, in order to deaf-termine the ideal number of amplifications for the test. DNA bands of 210 base pairs were observed after 25 and 30 cycles. From this result, 30 cycles was considered as suitable for the test.
For evaluation of sensitivity of the RT-PCR, the 104,8 TCID50/ml dengue type 2 CEA 2462 virus seed was tested at serial tenfold dilutions. Ampli-fied 210 base pair DNA bands were observed at 10-2 (weak band), 10-1, and 10-0 dilutions (Fig. 1). DNA amplification was not observed in some
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assays when undiluted virus seeds were processed, though a clear DNA amplification was obtained with the same virus seed at 10-1 dilution (Fig. 2).
Specific DNA amplification was observed with dengue type 2 CEA 2462, SPH 125367 and TOC 213 at 10-1 dilution. DNA amplification was not observed with yellow fever virus (Fig. 2).
DISCUSSION
The dengue type 2 specific primers utilized in this assay have been previously studied (Tanaka 1993, Morita et al. 1993, 1994). The high specific-ity of the 210 base pair sequence of the envelope protein gene produced in this study was exhaus-tively confirmed in the papers cited (Tanaka 1993, Morita et al. 1993, 1994) eliminating the need for further confirmation by hybridization or nested PCR. Also, the absence of DNA amplification of yellow fever virus excludes the possibility of any nonspecific amplification related to the virus, the cell culture or the nutrient medium in our study. Three dengue type 2 virus seeds obtained from cells which had confirmed infection by immunofluores-cence, were amplified by RT-PCR, showing a good correlation between these techniques.
Two-step PCR cycles have been reported as generating as much DNA as three-step cycles, at least for fragments about 200 base pairs long or less (Cha & Thilly 1995). A suitable extension of DNA in two-step PCR is accomplished by the high processivity of taqDNA polymerase action which occurs during the temperature transition of 53oC to 92oC. The optimal Taq DNA polymerase reac-tion range temperature from 70 to 75oC, took 15 sec in the cycles of our RT-PCR (Cha & Thilly 1995).
The 30 cycles of RT-PCR gave a very high sen-sitivity and a suitable amplification efficiency, since virus titers as low as 102,8 TCID50/ml could be detected. Virus titers in the blood during the first 6 days of dengue infection are commonly higher than this level, so the presence of the virus could be detected by RT-PCR (Gubler et al. 1981). However, in some tests, DNA amplification was not observed when undiluted virus seeds were pro-cessed, despite a clear DNA amplification obtained with the same virus seed at 10-1 dilution. Water used as sample diluent in our RT-PCR probably reduces RNAse, reverse transcriptase or DNA poly-merase inhibitors from the C6/36 cells, or reduces RNA excess present in straight samples inhibiting reverse transcriptase.
Compared to other reported assays (Shibata et al. 1990, Laille et al. 1991, Lanciotti et al. 1992, Tanaka 1993, Morita et al. 1993, 1994, Beaty et al. 1995) our RT-PCR shows the following advan-tages: (i) solubilization of the virus by detergent mixture (Nonidet P-40 and RNAse inhibitor) prior to the RT-PCR, avoids laborious extraction of vi-rus RNA especially when assaying many samples; (ii) the combination of RT and PCR which can be carried out after single addition of reagents in a single reaction vessel, reduces assay time, facili-tates the performance and reduces the risk of
con-Fig. 1: photograph of an agarose gel stained with ethidium bromide, showing 210 DNA base pair bands obtained from a tenfold serial dilution for titration of dengue type 2 CEA-2462 amplified by reverse transcription-polymerase chain reaction (RT-PCR).
NC: negative control (RT-PCR product of yellow fever virus).
MW 10-0 10-1 10-2 10-3 NC
200
MW CE TO SP NC
210
Fig. 2: photograph of an agarose gel stained with ethidium bromide, showing the specific DNA amplification by reverse transcription-polymerase chain reaction of the three dengue type 2 virus seeds at 10-1 dilution. Yellow fever virus was used as a negative control.
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398 Simple RT-PCR for Dengue Identification • LTM Figueiredo et al.
tamination; (iii) the use of small amount of reagents (1U of Taq DNA polimerase, 7.5 U of reverse tran-scriptase, 50 pmol of the primer pair) indicates that the method is reasonably economical. This makes the technique applicable even in the third world countries such as Brazil; (iiii) the two-step PCR cycles produce a clear DNA amplification, saves assay time and simplify the technique.
An obvious application of this RT-PCR is for confirming dengue type 2 virus isolation from pa-tients or mosquitoes, in tissue culture fluids. We intend to extend the use of this technique to other dengue serotypes, and, since dengue outbreaks presently occur in Brazil, we will test the amplifi-cation of dengue virus genome from clinical speci-mens.
ACKNOWLEDGEMENTS
To Dr Benedito Fonseca for helpful suggestions, Mrs Rita Helena Carlucci for technical assistance, and Dr David DeJong for review of the manuscript.
REFERENCES
Beaty BJ, Calisher CH, Shope RE 1995. Arboviruses, p. 189-212. In EH Lennette, DA Lennete, ET Lennete (eds) Diagnostic procedures for viral, rick-ettsial, and chlamydial infections, 7th ed., Ameri-can Public Health Association, Washington, D.C. Cha RS, Thilly WG 1995. Specificity, efficiency, and
fidelity of PCR, p. 37-52. In CW Dieffenbach, GS Dveksler (eds) PCR primer a laboratory manual. Cold Spring Harbor, New York.
Deubel VR, Kinney RM, Trent DW 1988. Nucleotide sequence and deduced amino acid sequence of the nonstructural proteins of Dengue type 2 virus, Ja-maica genotype: comparative analysis of the full lenght genome. Virology 165: 234-244.
Figueiredo LTM, Shope RE 1987. An enzyme immu-noassay for dengue antibody using infected cultured cells as antigen. J Virol Method 17: 191-198. Figueiredo LTM, Owa MA, Carlucci RH, Mello NV,
Fabbro AL, Capuano DM, Santili MB 1995. Den-gue serologic survey in Ribeirão Preto, SP, Brazil.
Bull PAHO 29: 59-69.
Gubler DJ 1987. Current research on dengue, p. 623-630. In Current Topics in Vector Research. Springer-Verlag, New York.
Gubler DJ, Suharyono W, Tan R, Abidin M, Sie A 1981. Viraemia in patients with naturally acquired dengue infection. Bull WHO 59: 623-630.
Henchal EA, Mc Cown JM, Seguin MK, Gentry MK, Brandt WE 1983. Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay. Am J Trop Med Hyg 32: 164-169.
Laille M, Deubel V, Sainte-Marie FF 1991. Demonstra-tion of concurrent dengue 1 and dengue 3 infecDemonstra-tion in six patients by the polimerase chain reaction. J Med Virol 34: 51-54.
Lanciotti RS, Calisher CH, Gubler DJ, Chang G-J, Vorndan AV 1992. Rapid detection and typing of dengue viruses from clinical samples by using re-verse transcriptase-polymerase chain reaction. J Clin Microbiol 30: 545-551.
Morita K, Tanaka M, Igarashi A 1993. Rapid identifi-cation of dengue virus serotypes by using polymerase chain reaction. J Clin Microbiol 29: 2107-2110. Morita K, Maemoto T, Honda S, Onishi K, Murata M,
Tanaka M, Igarashi A 1994. Rapid detection of vi-rus genome from imported dengue fever and den-gue hemorrhagic fever patients by direct polymerase chain reaction. J Med Virol 44: 54-58.
Reed LJ, Muench H 1938. A simple method of esti-mating fifty per cent end-points. Am J Hyg 27: 493-497
Shibata M, Kudo T, Kajiyama M, Takahashi T, Matsushima M, Kimura H, Kuzushima K, Morishita T 1990. Hepatitis C viral sequences in sera during the acute phase of infection. Am J Med 89: 830-832. Tanaka M 1993. Rapid identification of flavivirus using the polymerase chain reaction. J Virol Method 41: 311-322.
Wengler G 1991. Family Flaviviridae. In RIB Francki, Classification and nomenclature of viruses - Fifth report of the International Committee on Taxonomy of Viruses. Arch Virol (Suppl. 2): 230-231. WHO 1986. Dengue haemorrhagic fever: diagnosis,
